Background

Telomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.

Results

Significant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).

Conclusion

These results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.

Telomerase activity is detectable in 80–90% of malignancies and is absent in most normal somatic cells [1]. Breast cancer has been identified as an important target for developing telomerase inhibitors. Importantly, fine-needle aspirations of malignant breast tumors revealed that 81% were positive for telomerase [2], and Hiyama et al. (2000) also reported that 95% of advanced stage breast cancers express telomerase [3]. Due to the majority of tumor cells expressing telomerase, this protein is being evaluated as a tumor marker for breast cancer and other solid malignancies [4].

Telomerase, a ribonucleoprotein, synthesizes tandem repeats of the DNA sequence TTAGGG at the terminal ends of chromosomes permitting continuous genomic replication and cell division. Telomerase includes an RNA component (hTR) that serves as the template for telomeric DNA and a protein catalytic subunit (hTERT) with reverse transcriptase activity [5]. The activity of telomerase is regulated by the transcription of hTERT, and the cloning and characterization of the hTERT-promoter has permitted examination of elements controlling transcriptional activation and repression of hTERT [6].

To impact expression of hTERT in breast cancer cells, our research will utilize the E2F-1 transcription factor. The E2F family of transcription factors mediates cell cycle progression, and they are released upon phosphorylation of Retinoblastoma (Rb) family proteins [7–9]. While most of these factors are associated with inducing gene expression, E2F-1 has been identified as a transcriptional repressor of the hTERT gene, specifically binding to two separate sites (-174 bp and -98 bp) of the proximal hTERT promoter [7]. In addition, a study that examined the apoptotic function of E2F-1 found that cell death generated by ectopic expression of E2F-1 was independent of the p53 regulatory pathway [8]. Therefore, even in cells with intact p53 suppressor gene function, overexpression of E2F-1 should inhibit transcription of the hTERT gene and potentially induce apoptosis. Previous studies utilizing adenoviral-mediated transfection of E2F-1 genes induced apoptosis of melanoma cells [10], and an additional study revealed that E2F-1 suppressed cell growth while decreasing telomerase activity in the Tu-167 SCCHN cell line [11]. Adenoviral-mediated overexpression of E2F-1 also induced apoptosis in human breast and ovarian carcinoma cell lines independently of p53 [12]. We anticipate that telomerase repression via E2F-1 protein therapy could induce apoptotic activity and/or cell senescence in cancer cells more effectively than the viral-based studies described above.

Tat-mediated protein transduction was utilized in our research to effectively target cancer cells. Transduction occurs in a receptor- and transporter-independent fashion, targets the lipid bilayer, and also crosses the blood-brain barrier [13]. Tat has been reported to transduce 100% of mammalian cells, and studies also suggest the transcriptional effects of Tat-mediated protein transduction are reversible and have no detrimental effects because clearance of transduced proteins is dependent on the half-life of the protein [13–15]. Becker-Hapak et al. [14] also reported that Tat-mediated transduction is concentration dependent, reaches maximum intracellular concentration in less than 5 minutes, and nearly equal intracellular concentrations of the fusion proteins have been detected between all transduced cells.

Our research investigated primary infiltrating ductal carcinoma cells, both with and without a BRCA1 gene mutation. In 2001, published results by Ho et al. [16] determined that E2F-1 and E2F-4 were decreased in primary breast carcinomas, and 70% of the tumors revealed decreased expression of E2F-1. Additionally, the metastatic nodal tissue examined indicated that 100% of the tissue samples had significantly low levels of E2F-1 as compared to normal breast tissue. Ho et al. also suggested that E2Fs act as tumor suppressors in breast cancer and their down-regulation may be important in the development of metastases [16]. It is important to determine the repressive effects of E2F-1 on hTERT transcription in tumor cells with and without the normal BRCA1 protein to determine any variance between BRCA1 status and inhibition of telomerase. Recently, Wang et al. [17] determined that the BRCA1 promoter was transactivated in a dose-dependent manner by E2F-1 and served as a target for E2F-dependent transcriptional regulation. Other studies have also demonstrated that BRCA1 is a potent inducer of apoptosis by binding directly to p53 and stimulating transcriptional activation of pro-apoptotic genes such as Bax [18, 19]. Therefore, telomerase inhibition in the primary infiltrating ductal carcinoma cell line with a BRCA1 gene mutation, HCC1937, will be compared to a breast cancer cell line without detectible BRCA1 gene mutations, HCC1599. Aside from variance in BRCA1 status, both HCC1937 and HCC1599 cell lines carry similar mutations in p53, Her2/neu and ER/PR receptors [20].

Here we report the first results of the repressive effects of E2F-1 on telomerase activity using Tat-mediated transduction. These studies validated our proposed hypothesis that telomerase activity can be repressed in infiltrating ductal carcinoma cells via transcriptional regulation of hTERT utilizing protein transduction techniques with E2F-1 Tat fusion proteins.

We were able to successfully produce E2F-1/Tat fusion peptides utilizing both wildtype and mutant E2F-1 expression vectors to investigate their transcriptional effects on hTERT. Techniques were modified to produce both E2F-1/TatHA and E132/TatHA recombinant proteins (see Figure 1) and recombinant protein production was verified via western blotting. Protein transduction was determined in each cell line via immunocytochemistry, and the results supported effective transduction of E2F-1/TatHA, E132/TatHA and TatHA into all cell types. The TFPs were detected throughout the cell cytoplasm with perinuclear localization and detectable nuclear accumulation (Figure 2). The series of immunocytochemistry experiments ranging from 1 hour to 24 hours incubation with TFPs all revealed successful transduction of recombinant proteins into greater than 95% of carcinoma cells.

Our experiments were only performed with freshly dialyzed proteins to avoid protein precipitation and to ensure biological activity of proteins. The proteins were added to cells for 24 hours to assess differences in hTERT expression between the wildtype E2F1/TatHA protein, mutant E2F-1 (E132/TatHA) and the TatHA control protein. Following RNA isolation and real-time RT-qPCR, results revealed significant repression of hTERT in both HCC1937 and HCC1599 breast cancer cells. In HCC1937 cells, E2F-1/TatHA greatly repressed hTERT by 3.5-fold (p < 0.0024) (Figure 3) in comparison to the TatHA control proteins. Furthermore, the cells treated with E132/TatHA did not reveal effective repression of hTERT in comparison to TatHA (p < 0.6484).

In HCC1599 cells, a significant 3.3-fold repression of hTERT gene expression was observed with E2F-1/TatHA protein treatment versus the TatHA control protein (p < 0.0001) following 24 hours of treatment (Figure 3). Also, a slightly increased expression of hTERT (1.15-fold increase) was observed in TatHA treated HCC1599 cells versus the E132/TatHA protein, but this difference was only marginally statistically significant (p < 0.0102) (Figure 3). Further testing will be performed to assess differences between E132/TatHA and TatHA control proteins in larger in vitro test groups.

Minimal differences in hTERT expression were detected in HCC1937 and HCC1599 cells based on the BRCA1 status. As previously mentioned, the HCC1937 cell line has a homozygous BRCA1 mutation (5382C) in addition to mutations in p53, Her2/neu and ER/PR receptors. In contrast, the HCC1599 cell line expresses the wildtype BRCA1 tumor suppressor gene but also contains mutations in p53, Her2/neu and ER/PR receptors. A slightly higher repression of hTERT was observed in HCC1599 cells (4.0-fold) versus HCC1937 (3.5-fold) when E2F-1/TatHA was compared to the E132/TatHA control, but this slight difference in hTERT repression is most likely not attributed to the difference in BRCA1 status.

The results from this study suggest that E2F-1/TatHA is a moderately effective repressor of hTERT. In comparison to E132/TatHA and TatHA controls, hTERT was repressed by 3.3 to 4.0-fold in HCC1937 and HCC1599 breast carcinoma cells. The efficient transduction of the E2F-1/TatHA peptides into breast cancer cells combined with their transcriptional effects on hTERT expression suggests an alternate route to inhibit telomerase expression in breast cancer cells. The extent of hTERT repression in our study is comparable to the results observed by Crowe et al.'s study (2001) which suggests that our alternative route of introducing the E2F-1 transcription factor into cancer cells does not result in greater repression of hTERT than liposome-mediated transfection of E2F-1 [7]. However, the introduction of biologically active E2F-1/TatHA proteins has potential benefits. Treatment with our wildtype and mutant TFPs results in reversible repression of hTERT which could serve as a modality to augment the effects of other chemotherapeutic or apoptotic agents. This particular study did not examine the direct effects on apoptosis, but continued treatment with E2F-1/TatHA proteins could serve to effectively repress hTERT leading to long-term telomerase repression.

To interpret the results of our hTERT repression studies, it is important to review the expected effects of E2F-1/TatHA treatment of cells versus E132/TatHA and control peptide TatHA. Crowe et al. determined the normal E2F-1 expression vector binds to two sites of the hTERT promoter and represses its transcription by approximately 4-fold in their study [7]. The negative control, mutant E2F-1, referred to as E132/TatHA in our research, has a point mutation in the DNA-binding domain and cannot bind to the E2F-1 binding sites on the hTERT promoter and exhibited no repressive effects on hTERT [7]. We expected to observe similar results in our study, and since Tat-mediated protein transduction is an effective method of delivering biologically active proteins into cells, we hoped to achieve greater than 4.0-fold repression of hTERT in our study. However, as demonstrated by our data following 24 hours of treatment with E2F-1/TatHA, only a maximum of 4-fold hTERT repression was observed in HCC1599 cells.

The characterization of E2F-1 as a transcriptional repressor is a controversial role for this factor because it has traditionally been denoted as a transactivator of various genes. Hsieh et al. [21] reported that transcriptional repression rather than the transactivation function of E2F-1 is involved in the induction of apoptosis. Zhang et al. [19] provided the first direct evidence that telomerase is required for maintaining the vitality of both human tumor and immortal cells. Zhang et al. also reported that cell death in immortal cells was telomere-length dependent, and upon inhibiting telomerase in cells with short telomeres, the resulting damage to the chromosomes triggered apoptotic cell death [19]. Interestingly, Yamasaki et al., [22] determined that E2F-1 null mutant mice developed a variety of malignant tumors, and Field et al. [23] indicated that E2F-1 functions in mice to promote apoptosis and suppress cell proliferation. Tumor formation in the absence of E2F-1 was also suggested by Crowe et al. [7]. Their study utilized E2F-1 expression vectors and liposomal transfection into SCC25 cells resulting in transcriptional repression of hTERT via consensus binding in the hTERT- promoter at putative E2F-1 sites [7]. Importantly, Crowe et al. reported that transfection of the E2F-1 constructs reduced endogenous hTERT mRNA levels by 4-fold, and the reduced hTERT mRNA levels were accompanied by decreasing telomerase activity [7]. The study by Crowe et al. was one of the first to correlate the novel transcriptional repressive effects of E2F-1 on the hTERT gene promoter while monitoring subsequent telomerase activity [7]. However, the repressive effects of E2F-1 on hTERT must be further examined in future studies. The liposome-mediated transfection of E2F-1 via the Lipofectamine™ kit (Invitrogen Life Technologies, Carlsbad, CA) as described in Crowe et al. does not guarantee transfection of 100% of the cancer cells [7], nor do any of the adenoviral-mediated studies previously mentioned [10–12]. Therefore, to determine if E2F-1 can completely repress hTERT transcription, the E2F-1 protein must be effectively transduced into 100% of the cancer cells. Our research suggested greater than 95% transduction rates with E2F-1 TFPs, but maximum hTERT repression was only 4-fold with our alternative mode of transduction, which is comparable to results published by Crowe et al [7]. Complete repression of hTERT was not observed in our study using TFPs suggesting that a combination of transcriptional repressors may be required when using Tat-mediated transduction to completely inhibit expression of hTERT. It must be noted, that to date, complete gene repression has only been demonstrated utilizing gene knockout protocols and models. There are, however, significant advantages of using our method of transducing E2F-1 TFPs over transfection protocols. One is the simplicity of being able to use a TAT-peptide to transport any denatured protein inside the cell. Another is that it alleviates the deleterious effects associated with viral based DNA transfection procedures and does not result in permanent genetic manipulation. In addition, therapeutic treatment with these TFPs is completely reversible, permitting transient repression of target genes.

In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA and the TatHA peptide controls. In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control. A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control. No differences were observed in cell lines with wildtype versus mutant BRCA1 genes. Overall, these results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is a moderately effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599. The hypothesized repression of telomerase activity in infiltrating ductal carcinoma cells via E2F-1 repressor proteins is supported by significant hTERT repression in both HCC1937 and HCC1599 cells.

The breast cancer cell lines utilized in our study represent non-metastatic primary ductal carcinoma cell lines. HCC1937 cells were derived from a TNM stage IIB, grade 3 breast tumor, and the HCC1599 cell line was started from a TNM stage IIIA, grade 3 primary ductal carcinoma [20]. Our research suggests that E2F-1/TatHA is an effective repressor of hTERT in primary ductal carcinoma cells that are actively expressing telomerase. Although we have not fully investigated metastatic breast carcinoma cell lines, preliminary studies (results not shown) with MDA-MB-231 and MDA-MB-435S cells, both of which are derived from pleural effusions from metastatic breast cancer (TNM stage IV) [17], did not reveal statistically significant repression of hTERT. Therefore, the E2F-1/TatHA proteins investigated in this study would be potential adjuvants to therapy in nonmetastatic, primary breast tumors.

Collectively, these studies were undertaken to develop an effective technique to achieve complete transduction of cancer cells with fusion peptides. The hypothesized repression of telomerase activity in infiltrating ductal carcinoma cells via E2F-1 repressor proteins is supported by significant hTERT repression in both HCC1937 and HCC1599 cells. Future experiments will further investigate the transcriptional repressive effects of E2F-1 and its effects on cell-cycle progression and telomerase repression. To conclude, this therapy can initiate a sufficient biological effect to induce cell death in cancer cells but is expected to be safe enough to permit normal cells to recover from treatment without genetic alteration. Further research with E2F-1 TFPs will continue to explore their therapeutic potentials and benefits in the field of breast cancer research.

Western blotting

For Western blotting experiments, duplicate gels were run simultaneously- one gel for western blotting and a second gel for Coomassie blue staining. The gel designated for western blotting was transferred to nylon membranes using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's instructions using 1× TBS-T transfer buffer. Recombinant protein production was verified using primary mouse monoclonal anti-HA antibodies (1:1,000 dilution) (CRP, Inc., Denver, PA) and secondary HRP-labeled goat anti-mouse antibodies (1:25,000 dilution) (KPL, Inc., Gaithersburg, MD). Membranes were blocked with TBS-T/5% milk solution for 1 hour, incubated with primary anti-HA antibody for 1 hour, washed 3 times for 10 minutes each, and incubated with secondary HRP antibody for 1 hour. Following additional washings (3 × 10 minutes), western blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) according to manufacturer's instructions. Blots were exposed to X-ray film and developed.

Large-scale recombinant protein production

Recombinant E2F-1/TatHA and E132/TatHA proteins were purified under denaturing conditions with affinity chromatography using a modified procedure from Amersham Pharmacia [26]. Single colonies from protein expressing cultures inoculated 200 ml of 2YT broth supplemented with ampicillin (50 μg/ml) and were incubated at 37°C while shaking at 225 rpm for 16–18 hours. An additional 300 ml of broth was added along with fresh ampicillin and IPTG (400 μM), and cultures incubated an additional 6 hours. Cultures were centrifuged for 10 minutes at 6000 × g, and the pellets frozen at -20°C. Cell pellets were thawed for 20 minutes at 37°C, and 25 ml of Buffer 1 (20 mM Tris™-HCl, pH 8.0) added to resuspend the cells with vigorous pipetting. Cells were sonicated on ice (3 × 15 sec) and centrifuged (10 min × 6000 g). Cell pellets containing the inclusion bodies were then resuspended in 25 ml Buffer 2 (2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton™ X-100, pH 8.0). Samples were again sonicated on ice and centrifuged as described above. Pellets were resuspended in 25 ml Buffer 2 to wash the inclusion bodies and recentrifuged. A final wash of 25 ml of Buffer 1 was then performed, and following centrifugation, pellets were either used immediately or frozen at -20°C for a maximum of two weeks prior to use.

Cell culture experiments with recombinant proteins

HCC1937 and HCC1599 cells were seeded at a density of 2 × 105 cells/well in CellStar® 6-well tissue culture plates. HCC1937 (adherent) cells were allowed 24 hours to adhere to plates before recombinant proteins were added. HCC1599 (suspension) cells were seeded directly into wells with TFPs. After 24 hours of co-incubation, RNA was isolated and analyzed via RT-qPCR as described below.

RNA isolation, reverse transcription and quantitative PCR

Total RNA was isolated from cells using the Mini RNA Isolation II™ kit (Zymo Research, Orange, CA) using the following protocol. After treatment with TFPs, adherent cell lines were washed at least three times with 1× PBS and detached from tissue culture plates using 0.5 ml HyQ®tase™ (Hyclone, Logan, UT). Cells were added to 1.8 ml microcentrifuge tubes and pelleted by centrifugation at 14,000 rpm for 1 minute. Supernatant was decanted and excess fluid removed via micropipetting. A 600 μl volume of ZR RNA Buffer was added to cells, tubes vortexed 30 seconds, and sample transferred to a Zymo-Spin III column placed in a 2 ml collection tube. Zymo-Spin III column was centrifuged at 14,000 rpm for 1 minute. A 350 μl volume of RNA Wash Buffer was added to the Zymo-Spin III column and centrifuged as described above to wash column. A second wash was performed with 350 μl RNA Wash Buffer to further clean RNA. The spin column was then transferred to a sterile 1.8-ml microcentrifuge tube. A 50 μl volume of RNase-free water was added directly to the membrane of the Zymo-Spin III Column and column centrifuged for 15 seconds to elute RNA. The eluted RNA was quantified using a Nanodrop® ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE).

A two-step reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was performed for all the experiments described in this study. A 200 ng quantity of RNA was treated with DNase (1 U/μg) (Fisher Scientific, Pittsburgh, PA) in a 10 μl digestion reaction volume including a final 1× concentration of RQ1 RNase-Free DNase Reaction Buffer (40 mM Tris-HCl, 10 mM MgSO4, 1 mM CaCl2, pH 8.0) (Fisher Scientific, Pittsburgh, PA). Samples were incubated at 37°C for 30 minutes followed by the addition of 1 μl of RQ1 DNase Stop Solution (20 mM EGTA, pH 8.0) (Fisher Scientific, Pittsburgh, PA) and samples incubated at 65°C for 10 minutes to inactivate the DNase. The 11 μl volume of DNase-digested RNA was used for the RT-PCR reaction.

Real-time quantitative PCR

Real-time qPCR was performed using the MyiQ™ Single Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA). A 2 μl sample of each cDNA pool served as template for the qPCR reaction using IQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA) and 300 nM of each primer: hTERT sense & antisense primers (5'-GGAGCAAGTTGCAAAGCATTG-3' & 5'-CCCACGACGTAGTCCATGTT-3') [30], B-actin sense & antisense primers (5'-CACTCTTCCAGCCTTCCTTCC-3'& 5'-CTGTGTTGGCGTACAGGTCT-3') [31]. All qPCR reactions were performed in at least triplicate for each sample using 96-well plates. The following cycling program was used for all primer sets. Initial denaturation step at 95°C for 3 minutes followed by 45 cycles at 94°C- 30 sec., 59.5°C- 30 sec., 72°C- 30 sec., and an 80°C- 15 sec. data acquisition step. A melt curve was performed for every reaction plate. Fold changes in gene expression were determined using the 2-ΔΔCTmethod [32]. The CT data were imported into Microsoft Excel. CT values for hTERT and B-actin were averaged for each sample, and ΔCT calculated (ΔCT = CT, avg. hTERT – CT, avg. B-actin). ΔΔCT was calculated in the following manner: ΔΔCT = Avg. ΔCT, hTERT from E2F-1 or E132 TFP treatment – ΔCT, hTERT from TatHA control. Mean fold change in gene expression was determined by the following calculation: 2^-ΔΔCT.

Acknowledgements

We would like to thank Dr. Karen Vousdan and Dr. Steven Dowdy for providing vectors utilized in this research. We also would like to acknowledge the sources of funding: Susan G. Komen Breast Cancer Foundation Dissertation Research Award, #DISS0201652, and the Utah State Agricultural Experiment Station. Approved as UAES publication 7839.

Authors' original submitted files for images

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Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

KE performed cloning experiments, developed the protein purification protocols, developed real-time qRT-PCR experiments and experimental design. KE also performed statistical analysis and drafted manuscript. LR was involved in experimental design, coordinated the experiments and manuscript preparation. ML assisted with protein purification, maintained cancer cell cultures and performed real-time qRT-PCR. All authors have read and approved the final manuscript.

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